Abstract:
Retroviral gene marking has been used successfully in preclinical and clinical transplantation settings. Highly sensitive techniques for vector insertion-site determination, such as linear amplification-mediated polymerase chain reaction (PCR) in conjunction with next-generation sequencing, have been introduced to assess the composition of gene-marked hematopoiesis at a single-cell level. Here we used these novel techniques for directly comparing clonal reconstitution kinetics in mice transplanted with bone-marrow-derived stem cells genetically marked with either a standard, spleen focus-forming virus long terminal repeat-driven gamma-retroviral, or a lentiviral self-inactivating vector containing an identical but internal spleen focus-forming virus-derived enhancer/promoter. We observed that the use of the lentiviral self-inactivating vector for gene marking was associated with a broader repertoire of differently marked hematopoietic clones. More importantly, we found a significantly higher probability of insertions in growth-promoting, clonal-dominance-associated genes in the spleen focus-forming virus-long terminal repeat-driven gamma-retroviral vector at later time points of analysis. Based on our data, we suggest that the combined use of linear amplification-mediated PCR and next-generation sequencing represents a potent tool for the analysis of clonal reconstitution kinetics in the context of gene marking with integrated vectors. At the same time, our findings prove that the use of multiple restriction enzymes for linear amplification-mediated PCR is indispensable to detect most or ideally all individual stem cell clones contributing to hematopoiesis. We have also found that techniques such as quantitative PCR can be helpful to retrospectively analyze reconstitution kinetics for individual hematopoietic stem cell clones. Finally, our results confirm the notion that marking with lentiviral self-inactivating vectors is associated with a lower risk of genotoxicity as compared with gamma-retroviral long terminal repeat vectors.